Contacts of ribosomal proteins with tRNAPhe and 16S RNA in analogs of the 30S initiation complex

Direct RNA-protein contacts have been studied by means of ultraviolet-induced (254 nm) cross-links inside complexes of NAcPhe-tRNAPhe, Phe-tRNAPhe and deacylated tRNAPhe with poly(U)-charged 30S subunit of Escherichia coli ribosome. In the first two complexes tRNA directly contacts with the similar sets of proteins (S4, S5, S7, S9/S11; S6 and S8 are found only in the second complex). These sets are similar to that in the fMet-tRNAfMet X 30S X mRNA complex, evidencing similar disposition of tRNAs in these three complexes. 16S RNA contacts in free 30S subunit mainly with proteins S4, S7 and S9/S11. In both complexes, containing NAcPhe-tRNAPhe and Phe-tRNAPhe, 16S RNA contacts with essentially the same proteins (S4, S5, S7, S8, S9/S11, S10, S15, S16 and S17) and in the same ratio, evidencing similar conformation of 30S subunit in these two complexes. In the third complex deacylated tRNAPhe contacts with proteins S4, S5, S6, S8, S9/S11 and S15, 16S RNA-protein interaction differs from those in the first two complexes by a remarkable decrease of cross-linked proteins S8, and S9/S11 and by the appearance of a large amount of cross-linked proteins(s) S13/S14. Hence, this complex differs from the first two by conformation of 30S subunit and, probably, by disposition and/or conformation of tRNA.     

Cotranscription and processing of 23S, 4.5S and 5S rRNA in chloroplasts from Zea mays

The termini of rRNA processing intermediates and of mature rRNA species encoded by the 3' terminal region of 23S rDNA, by 4.5S rDNA, by the 5' terminal region of 5S rDNA and by the 23S/4.5S/5S intergenic regions from Zea mays chloroplast DNA were determined by using total RNA isolated from maize chloroplasts and 32P-labelled rDNA restriction fragments of these regions for nuclease S1 and primer extension mapping. Several processing sites detectable by both 3' and 5' terminally labelled probes could be identified and correlated to the secondary structure for the 23S/4.5S intergenic region. The complete 4.5S/5S intergenic region can be reverse transcribed and a common processing site for maturation of 4.5S and 5S rRNA close to the 3' end of 4.5S rRNA was detected. It is therefore concluded that 23S, 4.5S and 5S rRNA are cotranscribed.     

Isolation of ribosome particles from meningopneumonitis organisms

In ribonucleic acid (RNA) extracted by phenol and sodium dodecyl sulfate from purified reticulate bodies of meningopneumonitis (MP) organisms, 21S, 16S, and 4S RNA were found by sucrose density gradient sedimentation analysis. When purified reticulate bodies were homogenized by sonic treatment or by treatment with sodium deoxycholate and were fractionated by differential centrifugation, more than 50% of the RNA was recovered in the fraction which was sedimented by centrifugation at 105,000 x g for 2 hr, but not at 13,000 x g for 20 min. From homogenates prepared in this manner, 50S and 30S particles containing RNA were isolated by sucrose density gradient centrifugation. These 50S and 30S particles were also found in lysates of cytoplasmic fractions of infected cells which were labeled by (32)P during 17 to 17.5 hr or 15 to 18 hr after infection. The synthesis of 50S and 30S particles was not inhibited by actinomycin D. When infected cells were homogenized in the presence of 0.01 or 0.02 m MgCl(2), 70S particles were isolated instead of 50S and 30S particles. When dialyzed against low concentrations of MgCl(2), the 70S particles dissociated to 50S and 30S particles. The base ratio of the 70S particles is very similar to that of 16S plus 21S RNA. The characteristics of the 70S, 50S, and 30S particles suggest that these are ribosome particles, similar to bacterial ribosomes.     

A comparative study on 40S ribosomal proteins of Artemia salina and rat liver: micro analysis of amino acid composition by high-performance liquid chromatography

The amino acid compositions of 24 proteins of 40S ribosomal subunits of Artemia salina cysts were determined and compared with those of rat liver. The basic proteins of A. salina 40S ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and extracted with 70% formic acid. Samples were freed from contaminants by gel-filtration through a high-performance liquid chromatography column. Amino acid compositions were determined for individual proteins by pre-column derivatization with N,N-dimethylaminoazobenzenesulfonyl chloride followed by reverse phase high-performance liquid chromatography. The similarity of amino acid compositions between A. salina and rat liver 40S ribosomal proteins was evaluated by the method of Cornish-Bowden (Cornish-Bowden, A. (1980) Anal. Biochem. 105, 233-238), and possible relationships between A. salina and rat were detected for 16 protein species (S2, S3, S4, S6, S7, S8, S15a, S16, S17, and S18, strongly related and S14, S15, S20, S23, S24, and S26, weakly related), indicating a conservative nature of eukaryotic ribosomal proteins.     

The distance between S1, S21, and the 3' end of 16S RNA in 30S ribosomal subunits. The effect of poly(uridylic acid) and 50S subunits on these distances

The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3' end of 16S RNA in Escherichia coli 30S ribosomal subunits were determined by non-radiative energy transfer to be: S21-16S RNA, 5.1 nm; S21-S1, 6.9 nm; S1-16S RNA, 6.8 nm. Binding of poly(uridylic acid) to 30S subunits causes the apparent distances between S1 and 16S RNA or S21 and 16S RNA to increase by more than 1.2 nm and 0.5 nm, respectively, but has little or no effect on the S1-S21 distance. Binding of 50S subunits causes an apparent increase in the S21-16S RNA and S21-S1 distances by 1.0 nm and 0.8 nm, respectively, but has little or no effect on the S1-16S-RNA distance.     

Interaction of ribosomal proteins S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA from Escherichia coli

The co-operative interaction of 30 S ribosomal subunit proteins S6, S8, S15 and S18 with 16 S ribosomal RNA from Escherichia coli was studied by (1) determining how the binding of each protein is influenced by the others and (2) characterizing a series of protein-rRNA fragment complexes. Whereas S8 and S15 are known to associate independently with the 16 S rRNA, binding of S18 depended upon S8 and S15, and binding of S6 was found to require S8, S15 and S18. Ribonucleoprotein (RNP) fragments were derived from the S8-, S8/S15- and S6/S8/S15/S18-16 S rRNA complexes by partial RNase hydrolysis and isolated by electrophoresis through Mg2+-containing polyacrylamide gels or by centrifugation through sucrose gradients. Identification of the proteins associated with each RNP by gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of S8, S8 + S15 and S6 + S8 + S15 + S18 in the corresponding fragment complexes. Analysis of the rRNA components of the RNP particles confirmed that S8 was bound to nucleotides 583 to 605 and 624 to 653, and that S8 and S15 were associated with nucleotides 583 to 605, 624 to 672 and 733 to 757. Proteins S6, S8, S15 and S18 were shown to protect nucleotides 563 to 605, 624 to 680, 702 to 770, 818 to 839 and 844 to 891, which span the entire central domain of the 16 S rRNA molecule (nucleotides 560 to 890). The binding site for each protein contains helical elements as well as single-stranded internal loops ranging in size from a single bulged nucleotide to 20 bases. Three terminal loops and one stem-loop structure within the central domain of the 16 S rRNA were not protected in the four-protein complex. Interestingly, bases within or very close to these unprotected regions have been shown to be accessible to chemical and enzymatic probes in 30 S subunits but not in 70 S ribosomes. Furthermore, nucleotides adjacent to one of the unprotected loops have been cross-linked to a region near the 3' end of 16 S rRNA. Our observations and those of others suggest that the bases in this domain that are not sequestered by interactions with S6, S8, S15 or S18 play a role involved in subunit association or in tertiary interactions between portions of the rRNA chain that are distant from one-another in the primary structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of proteins S1, S2, S16 and S23 on the surface of small subunits of rat liver ribosomes by immune electron microscopy

Summary Ribosomal proteins S1, S2, S16 and S23 were localized on the surface of the small subunit (40S) of rat liver ribosomes by immune electron microscopy. Antibodies against the single proteins were raised in rabbits and chicken and purified by affinity chromatography. 40S-IgG-40S complexes were obtained by incubation of 40S subunits with non-crossreacting antibodies specific for each of the four proteins and subsequent sucrose density gradient centrifugation. The location of the proteins was determined by means of antibody binding sites visualized in negative contrast in the electron microscope. The four investigated proteins are mainly located in the head region of the small subunit. Exposed antigenic determinants of proteins S1 and S2 were found to be located at different sites of the small subunit whereas proteins S16 and S23 were mapped in a limited region only.S2,S3,S17,S21 according to the new nomenclature (McConkey et al., 1979)     

Pulsed-field gel electrophoresis of the genomic restriction fragments of coagulase-negative staphylococci

Abstract The genomes of 47 coagulase-negative staphylococcal strains assigned to different species were analysed by pulsed-field electrophoresis. The strains were clustered on the basis of their similarity in the Sma I restriction patterns into various groups, each group consisting of the type strain and the strains whose Sma I restriction patterns were similar to that of the type strain. The Sma I restriction groups seem to correspond to the following species: Staphylococcus warneri, S. hominis, S. xylosus, S. lugdunensi, S. kloosii, S. haemolyticus, S. lentus, S. cohnii, S. equorum, S. chromogenes, S. saprophyticus, S. simulans, S. carnosus, S. capitis and S. auricularis . The species S. sciuri, S. caseolyticus, S. gallinarum, S. epidermidis and S. schleiferi were represented only by their type strains and showed no similarity in their Sma I restriction patterns neither to each other nor to all the species investigated here. Thus, the classification of coagulase-negative staphylococcal strains into the above species seems to be confirmed also by genome restriction analysis carried out by pulsed-field gel electrophoresis.     

Cross-linking study on protein neighborhoods at the subunit interface of rat liver ribosomes with 2-iminothiolane

Rat liver 80 S ribosomes were cross-linked with 2-iminothiolane. Proteins extracted from the cross-linked 80 S ribosomes were separated into 25 fractions by chromatography on carboxy methylcellulose. Each protein fraction was analyzed by diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Eight pairs characteristic of 80 S ribosomes were detected which did not appear when isolated 40 S and 60 S subunits were cross-linked, and the cross-linked proteins were analyzed in similar manners. The cross-linked components were radioiodinated and then analyzed by two-dimensional gel electrophoresis, followed by autoradiography. Eight kinds of cross-links between 60 S subunit proteins and 40 S subunit proteins were identified as follows: SA30 (acidic protein with Mr 30,000)-LA33 (acidic protein with Mr 33,000), S2-LA33, S2-L11, S3a-L11, S4-L5, S25-L5, S4-L24 and S6-L24.     

Central domain assembly: thermodynamics and kinetics of S6 and S18 binding to an S15-RNA complex

The 30 S ribosomal subunit assembles in vitro through the hierarchical binding of 21 ribosomal proteins to 16 S rRNA. The central domain of 16 S rRNA becomes the platform of the 30 S subunit upon binding of ribosomal proteins S6, S8, S11, S15, S18 and S21. The assembly of the platform is nucleated by binding of S15 to 16 S rRNA, followed by the cooperative binding of S6 and S18. The prior binding of S6 and S18 is required for binding of S11 and S21. We have studied the mechanism of the cooperative binding of S6 and S18 to the S15-rRNA complex by isothermal titration calorimetry and gel mobility shift assays with rRNA and proteins from the hyperthermophilic bacterium Aquifex aeolicus. S6 and S18 form a stable heterodimer in solution with an apparent dissociation constant of 8.7 nM at 40 degrees C. The S6:S18 heterodimer binds to the S15-rRNA complex with an equilibrium dissociation constant of 2.7 nM at 40 degrees C. Consistent with previous studies using rRNA and proteins from Escherichia coli, we observed no binding of S6 or S18 in the absence of the other protein or S15. The presence of S15 increases the affinity of S6:S18 for the RNA by at least four orders of magnitude. The kinetics of S6:S18 binding to the S15-rRNA complex are slow, with an apparent bimolecular rate constant of 8.0 x 10(4) M(-1) s(-1) and an apparent unimolecular dissociation rate of 1.6 x 10(-4) s(-1). These results, which are consistent with a model in which S6 and S18 bind as a heterodimer to the S15-rRNA complex, provide a mechanistic framework to describe the previously observed S15-mediated cooperative binding of S6 and S18 in the ordered assembly of a multi-protein ribonucleoprotein complex.     

Biogenesis of mitochondria. 53

Summary As shown by gel electrophoresis analysis, E. coli mutant 219 is mutated on the gene coding for S4. This mutant and the parental strain have been studied at the permissive (30°) and the non-permissive temperature (42°) for ribosome assembly and r-protein biosynthesis.The extracts of cells grown at the non-permissive temperature were analyzed by sucrose gradients: Particles sedimenting more slowly (28S) than normal 30S accumulate while 50S precursors undergo maturation and attach to the preformed 30S subunits yielding 70S ribosomes. In addition a small but detectable amount of 30S is also synthesized at 42°. The 28S particles contain all 30S r-proteins except S1, S2 and S12; S5, S7 and S21 are present in reduced amount.The relative rate of biosynthesis of individual r-proteins was determined by pulse-labelling the cells with radioactive leucine. Individual r-proteins were purified from cell extract by the three-dimensional gel electrophoresis technique. The relative rate of biosynthesis of 50S proteins is unchanged in mutant cells grown at 42°. Only the rate of synthesis of five 30S proteins is modified by the temperature shift: S10, S13, S20 and S21 have an increased rate, while S18 is synthesized at a reduced rate. Thus in cells deficient in the assembly of 30S subunits, although the biosynthesis of a few 30S r-proteins is specifically altered, the synthesis of most r-proteins appears to be controlled in the same way as are total cell proteins.     

Changes in Acetylcholinesterase Molecular Forms During the Embryonic Development of Torpedo marmorata

Abstract: Multiple molecular forms of acetylcholinesterase from electric organ and electric lobe of Torpedo marmorata were examined at various developmental stages by sucrose density sedimentation. Four major forms were characterized by their apparent sedimentation coefficients of 6 S, 11 S, 13 S, and 17 S. Embryonic lobe possessed at early stages predominantly the 11 S form. With maturation the 17 S form became the most abundant. The early embryonic stages of the electric organ were characterized by predominating amounts of 6 S and 11 S forms. With differentiation of the postsynaptic membrane of the developing electrocytes, 13 S and 17 S forms replaced the slower-sedimenting forms. Concomitant with the formation of synaptic contacts, a transient increase in the 13 S form was followed by a dramatic accumulation of rapid-sedimenting 17 S form. The establishment of fully functional synapses was accompanied by an increase in the amount of the hydrophobic 6 S form. At birth, equal amounts of 6 S and 17 S form were found, with the other forms present in only trace amounts. The observed characteristic changes correlated with morphological and physiological events, indicating a close functional relationship between the accumulation of the 17 S form and synapse formation and the accumulation of the 6 S form and onset of function.     

Function and stability of human transcobalamin II: role of intramolecular disulfide bonds C98-C291 and C147-C187

The current studies have investigated the role of three disulfide bonds of human transcobalamin II (TC II), a plasma transporter of cobalamin (Cbl; vitamin B12), in its function and stability. When translated in vitro in the presence or absence of microsomal vesicles, TC II constructs with a single substitution, C3S or C249S, demonstrated synthesis of a stable functional protein. However, TC II synthesized in the presence of microsomal vesicles using constructs with a single (C98S, C147S, C187S, C291S), double (C3/147/S, C98/147/S) or triple (C3/98/147/S) substitution was unstable. In the absence of microsomal vesicles, the percentage of binding to Cbl-Sepharose matrix by TC II expressed by constructs C3S, C3/147/S, C98/147/S, or C3/98/147/S was 100, 49, 52, and 35%, respectively. Upon their reductive alkylation, the binding of TC II expressed by these constructs was reduced to approximately 25-30%. TC II constructs C3S or C249S, when expressed in TC II-deficient fibroblasts, produced a stable functional protein, but those expressed by constructs C147S, C187S, C291S, C3/147/S, C98/147/S, or C3/98/147/S were rapidly degraded. The intracellular degradation of TC II expressed by these constructs was inhibited by lactacystin or MG-132 but not by the lysosomal degradation inhibitors ammonium chloride or chloroquine. These studies suggest that optimal binding of Cbl by human TC II is supported by disulfide bonds C98-C291 and C147-C187 and that their disruption results in loss of Cbl binding and their rapid degradation by the proteasomal machinery.     

Regulation of ribosomal S6 kinase 2 by effectors of the phosphoinositide 3-kinase pathway

Ribosomal S6 kinase (S6K1), through phosphorylation of the 40 S ribosomal protein S6 and regulation of 5'-terminal oligopyrimidine tract mRNAs, is an important regulator of cellular translational capacity. S6K1 has also been implicated in regulation of cell size. We have recently identified S6K2, a homolog of S6K1, which phosphorylates S6 in vitro and is regulated by the phosphatidylinositide 3-kinase (PI3-K) and mammalian target of rapamycin pathways in vivo. Here, we characterize S6K2 regulation by PI3-K signaling intermediates and compare its regulation to that of S6K1. We report that S6K2 is activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and protein kinase Czeta but that S6K2 is more sensitive to basal activation by myristoylated protein kinase Czeta than is S6K1. The C-terminal sequence of S6K2 is divergent from that of S6K1. We find that the S6K2 C terminus plays a greater role in S6K2 regulation than does the S6K1 C terminus by functioning as a potent inhibitor of activation by various agonists. Removal of the S6K2 C terminus results in an enzyme that is hypersensitive to agonist-dependent activation. These data suggest that S6K1 and S6K2 are similarly activated by PI3-K effectors but that sequences unique to S6K2 contribute to stronger inhibition of its kinase activity. Understanding the regulation of the two S6K homologs may provide insight into the physiological roles of these kinases.     

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.     

Characterization of Nucleic Acid of Pichinde Virus

The nucleic acid of Pichinde virus was found to be single-stranded ribonucleic acid (RNA) as determined by sensitivity to ribonuclease, by alkaline degradation, by buoyant density in cesium sulfate, and by analysis of the base composition. The RNA of the virion could be separated into five components which had sedimentation coefficients corresponding to 31S, 28S, 22S, 18S and 4 to 6S. The 28S, 18S, and possibly the 4 to 6S RNAs appear to be derived from host cell components incorporated into the virion, whereas the 31S and 22S components appear to represent the genome of the virus.     

Purification and properties of dyneins from Paramecium cilia

Dynein ATPases were purified from Paramecium cilia by salt extraction followed by sucrose density gradient centrifugation and anion exchange chromatography. The two major dyneins sedimented in sucrose gradients as species of 22 S and 12 S. After purification by anion exchange chromatography, their specific activities were about 0.4 and 0.5 mumol/min per mg, respectively. The dyneins could be distinguished by subunit composition and immunological crossreactivity. Sucrose density gradient centrifugation revealed additional ATPase activity in the region between the 22 S and 12 S dyneins, including a 19 S activity. Mg2+-ATPase activities of the dyneins and the 19 S activity were inhibited by vanadate and Zn2+, and were activated by Triton X-100. Antibodies against the 22 S dynein from Paramecium reacted on immunoblots with most of the polypeptides of 22 S dynein, and showed that the heavy chains of 22 S dynein are not identical to those that sediment at 19 S and 12 S. Several minor ATPase activities were revealed by anion exchange chromatography of fractions from the 22 S, 19 S and 12 S regions of sucrose gradients. These minor activities were stimulated by Mg2+, inhibited by vanadate, and could be distinguished from each other by their elution positions and polypeptide compositions.     

The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

Glycogen synthase kinase 3beta phosphorylates Alzheimer's disease-specific Ser396 of microtubule-associated protein tau by a sequential mechanism

Phosphorylation of tau on S(396) was suggested to be a key step in the development of neurofibrillary pathology in Alzheimer's disease brain [Bramblett, G. T., Goedert, M., Jacks, R., Merrick, S. E., Trojanowski, J. Q., and Lee, V. M.-Y. (1993) Neuron 10, 1089-1099]. GSK3beta phosphorylates Ser(396) of tau in the brain by a mechanism which is not clear. In this study, when HEK-293 cells were cotransfected with tau and GSK3beta, GSK3beta co-immunoprecipitated with tau and phosphorylated tau on S(202), T(231), S(396), and S(400) but not on S(262), S(235), and S(404). Blocking phosphorylation on T(231), S(235), S(396), S(400), or S(404) did not prevent the subsequent phosphorylation on S(202) by GSK3beta. These data suggest that GSK3beta directly phosphorylates tau on S(202) (without requiring prephosphorylation). However, preventing phosphorylation on S(235), S(400), and S(404) prevented GSK3beta-dependent phosphorylation of T(231), S(396), and S(400), respectively. This indicates that phosphorylation of T(231), S(396), and S(400) by GSK3beta depends on a previous phosphorylation of S(235), S(400), and S(404), respectively. To examine S(396) phosphorylation, we analyzed phosphorylation of S(396), S(400), and S(404). Blocking phosphorylation of S(404) prevented the subsequent GSK3beta-dependent phosphorylation of both S(400) and S(396). When phosphorylation of S(404) was allowed but S(400) blocked, GSK3beta failed to phosphorylate S(396). Thus, GSK3beta phosphorylates S(396) by a two-step mechanism. In the first step, GSK3beta phosphorylates S(400) of previously S(404)-phosphorylated tau. This event primes tau for second-step phosphorylation of S(396) by GSK3beta. We conclude that GSK3beta phosphorylates tau directly at S(202) but requires the previous phosphorylation on S(235) to phosphorylate T(231). Phosphorylation of S(396), on the other hand, occurs sequentially. Once a priming kinase phosphorylates S(404), GSK3beta sequentially phosphorylates S(400) and then S(396).     

A method of preparing Escherichia coli 16 S RNA possessing previously unobserved 30 S ribosomal protein binding sites

A method of preparing 16 S RNA has been developed which yields RNA capable of binding specifically at least 12, and possibly 13, 30 S ribosomal proteins. This RNA, prepared by precipitation from 30 S subunits using a mixture of acetic acid and urea, is able to form stable complexes with proteins S3, S5, S9, S12, S13, S18 and possibly S11. In addition, this RNA has not been impaired in its capacity to interact with proteins S4, S7, S8, S15, S17 and S20, which are proteins that most other workers have shown to bind RNA prepared by the traditional phenol extraction procedure (Held et al., 1974; Garrett et al., 1971; Schaup et al., 1970,1971).We have applied several criteria of specificity to the binding of proteins to 16 S RNA prepared by the acetic acid-urea method. First, the new set of proteins interacts only with acetic acid-urea 16 S RNA and not with 16 S RNA prepared by the phenol method or with 23 S RNA prepared by the acetic acid-urea procedure. Second, 50 S ribosomal proteins do not interact with acetic acidurea 16 S RNA but do bind to 23 S RNA. Third, in the case of protein S9, we have shown that the bound protein co-sediments with acetic acid-urea 16 S RNA in a sucrose gradient. Additionally, a saturation binding experiment showed that approximately one mole of protein S9 binds acetic acid-urea 16 S RNA at saturation. Thus, we conclude that the method employed for the preparation of 16 S RNA greatly influences the ability of the RNA to form specific protein complexes. The significance of these results is discussed with regard to the in vitro assembly sequence.